Acellular pertussis vaccines and methods of preparation thereof

ABSTRACT

Acellular pertussis vaccines comprise purified toxin or toxoid thereof, filamentous haemagglutinin, pertactin and fimbrial agglutinogens formulated to confer protection to at least 70% of members of an at-risk population. The fimbrial agglutinogens may be prepared from a Bordetella strain, particularly a  B. pertussis  strain, by a multiple step procedure involving extraction of the fimbrial agglutinogens from cell paste and concentrating and purifying the extracted material.

REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part of copending U.S.patent application Ser. No. 08/501,743 filed Jul. 12, 1995, which itselfis a continuation-in-part of copending U.S. patent application Ser. No.08/433,646 filed May 4, 1995.

FIELD OF INVENTION

[0002] The present invention relates to acellular pertussis vaccines,components thereof, and their preparation.

BACKGROUND TO THE INVENTION

[0003] Whooping cough or pertussis is a severe, highly contagious upperrespiratory tract infection caused by Bordetella pertussis. The WorldHealth Organization estimates that there are 60 million cases ofpertussis per year and 0.5 to 1 million associated deaths (ref. 1.Throughout this specification., various references are referred to inparenthesis to more fully describe the state of the art to which thisinvention pertains. Full bibliographic information for each citation isfound at the end of the specification, immediately following the claims.The disclosures of these references are hereby incorporated by referenceinto the present disclosure) In unvaccinated populations, a pertussisincidence rate as high as 80% has been observed in children under 5years old (ref. 2). Although pertussis is generally considered to be achildhood disease, there is increasing evidence of clinical andasymptomatic disease in adolescents and adults (refs. 3, 4 and 5).

[0004] The introduction of whole-cell vaccines composed of chemically-and heat-inactivated B. pertussis organisms in the 1940's wasresponsible for a dramatic reduction in the incidence of whooping coughcaused by B. pertussis. The efficacy rates for whole-cell vaccines havebeen estimated at up to 95% depending on case definition (ref. 6) .While infection with B. pertussis confers life-long immunity, there isincreasing evidence for waning protection after immunization withwhole-cell vaccines (ref. 3). Several reports citing a relationshipbetween whole-cell pertussis vaccination, reactogenicity and seriousside-effects led to a decline in vaccine acceptance and consequentrenewed epidemics (ref. 7). More recently defined component pertussisvaccines have been developed.

Antigens for Defined Pertussis Vaccines

[0005] Various acellular pertussis vaccines have been developed andinclude the Bordetella pertussis antigens, Pertussis Toxin (PT),Filamentous haemagglutonin (FHA), the 69 kDa outer membrane protein(pertactin) and fimbrial agglutinogens (see Table 1 below. The Tablesappear at the end of the specification).

Pertussis Toxin

[0006] Pertussis toxin is an exotoxin which is a member of the A/Bfamily of bacterial toxins with ADP-ribosyltransferase activity (ref.8). The A-moiety of these toxins exhibit the ADP-ribosyltransferaseactivity and the B portion mediates binding of the toxin to host cellreceptors and the translocation of A to its site of action. PT alsofacilitates the adherence of B. pertussis to ciliated epithelial cells(ref. 9) and also plays a role in the invasion of macrophages by B.pertussis (ref. 10).

[0007] All acellular pertussis vaccines have included PT, which has beenproposed as a major virulence factor and protective antigen (ref. 11,12). Natural infection with B. pertussis generates both humoral andcell-mediated responses to PT (refs. 13 to 17). Infants havetransplacentally-derived anti-PT antibodies (refs. 16, 18) and humancolostrum containing anti-PT antibodies was effective in the passiveprotection of mice against aerosol infection (ref. 19). A cell-mediatedimmune (CMI) response to PT subunits has been demonstrated afterimmunization with an acellular vaccine (ref. 20) and a CMI response toPT was generated after whole-cell vaccination (ref. 13).Chemically-inactivated PT in whole-cell or component vaccines isprotective in animal models and in humans (ref. 21) Furthermore,monoclonal antibodies specific for subunit S1 protect against B.pertussis infection (refs. 22 and 23).

[0008] The main pathophysiological effects of PT are due to itsADP-ribosyltransferase activity. PT catalyses the transfer of ADP-ribosefrom NAD to the G_(i) guanine nucleotide-binding protein, thusdisrupting the cellular adenylate cyclase regulatory system (ref. 24).PT also prevents the migration of macrophages and lymphocytes to sitesof inflammation and interferes with the neutrophil-mediated phagocytosisand killing of bacteria (ref. 25). A number of in vitro and in vivoassays have been used to asses the enzymatic activity of S1 and/or PT,including the ADP-ribosylation of bovine transducin (ref. 26), theChinese hamster ovary (CHO) cell clustering assay (ref. 27) , histaminesensitization (ref. 28), leukocytosis, and NAD glycohydrolase. Whenexposed to PT, CHO cells develop a characteristic clustered morphology.This phenomenon is dependent upon the binding of PT, and subsequenttranslocation and ADP-ribosyltransferase activity of S1 and thus the CHOcell clustering assay is widely used to test the integrity and toxicityof PT holotoxins.

Filamentous Haemagglutonin

[0009] Filamentous haemagglutonin is a large (220 kDa) non-toxicpolypeptide which mediates attachment of B. pertussis to ciliated cellsof the upper respiratory tract during bacterial colonization (refs. 9,29). Natural infection induces anti-FHA antibodies and cell mediatedimmunity (refs. 13, 15, 17, 30 and 31). Anti-FHA antibodies are found inhuman colostrum and are also transmitted transplacentally (refs. 17, 18and 19). Vaccination with whole-cell or acellular pertussis vaccinesgenerates anti-FHA antibodies and acellular vaccines containing FHA alsoinduce a CMI response to FHA (refs. 20, 32). FHA is a protective antigenin a mouse respiratory challenge model after active or passiveimmunization (refs. 33, 34). However, alone FHA does not protect in themouse intracerebral challenge potency assay (ref. 28).

69 kDa Outer Membrane Protein (Pertactin)

[0010] The 69 kDa protein is an outer membrane protein which wasoriginally identified from B. bronchiseptica (ref. 35). It was shown tobe a protective antigen against B. bronchiseptica and was subsequentlyidentified in both B. pertussis and B. parapertussis. The 69 kDa proteinbinds directly to eukaryotic cells (ref. 36) and natural infection withB. pertussis induces an anti-P.69 humoral response (ref. 14) and P.69also induces a cell-mediated immune response (ref. 17, 37, 38).Vaccination with whole-cell or acellular vaccines induces anti-P.69antibodies (refs. 32, 39) and acellular vaccines induce P.69 CMI (ref.39). Pertactin protects mice against aerosol challenge with B. pertussis(ref. 40) and in combination with FHA, protects in the intracerebralchallenge test against B. pertussis (ref. 41). Passive transfer ofpolyclonal or monoclonal anti-P.69 antibodies also protects mice againstaerosol challenge (ref. 42).

Agglutinogens

[0011] Serotypes of B. pertussis are defined by their agglutinatingfimbriae. The WHO recommends that whole-cell vaccines include types 1, 2and 3 agglutinogens (Aggs) since they are not cross-protective (ref.43). Agg 1 is non-fimbrial and is found on all B. pertussis strainswhile the serotype 2 and 3 Aggs are fimbrial. Natural infection orimmunization with whole-cell or acellular vaccines induces anti-Aggantibodies (refs. 15, 32). A specific cell-mediated immune response canbe generated in mice by Agg 2 and Agg 3 after aerosol infection (ref.17). Aggs 2 and 3 are protective in mice against respiratory challengeand human colostrum containing anti-agglutinogens will also protect inthis assay (refs. 19, 44, 45).

Acellular Vaccines

[0012] The first acellular vaccine developed was the two-componentPT+FHA vaccine (JNIH 6) of Sato et al. (ref. 46). This vaccine wasprepared by co-purification of PT and FHA antigens from the culturesupernatant of B. pertussis strain Tohama, followed by formalintoxoiding. Acellular vaccines from various manufacturers and of variouscompositions have been used successfully to immunize Japanese childrenagainst whopping cough since 1981 resulting in a dramatic decrease inincidence of disease (ref. 47). The JNIH 6 vaccine and a mono-componentPT toxoid vaccine (JNIH 7) were tested in a large clinical trial inSweden in 1986. Initial results indicated lower efficacy than thereported efficacy of a whole-cell vaccine, but follow-up studies haveshown it to be more effective against milder disease diagnosed byserological methods (refs. 48, 49, 50, 51). However, there was evidencefor reversion to toxicity of formalin-inactivated PT in these vaccines.These vaccines were also found to protect against disease rather thaninfection.

[0013] A number of new acellular pertussis vaccines are currently beingassessed which include combinations of PT, FHA, P.69, and/oragglutinogens and these are listed in Table 1. Several techniques ofchemical detoxication have been used for PT including inactivation withformalin (ref. 46), glutaraldehyde (ref. 52), hydrogen peroxide (ref.53), and tetranitromethane (ref. 54).

[0014] Thus, current commercially-available acellular pertussis vaccinesmay not contain appropriate formulations of appropriate antigens inappropriate immunogenic forms to achieve a desired level of efficacy ina pertussis-susceptible human population.

[0015] It would be desirable to provide efficacious accellular pertussisvaccines containing selected relative amounts of selected antigens andmethods of production thereof.

SUMMARY OF THE INVENTION

[0016] The present invention is directed towards acellular pertussisvaccine preparations, components thereof, methods of preparation of suchvaccines and their components, and methods of use thereof.

[0017] In a further aspect of the invention, there is provided animmunogenic composition comprising the fimbrial agglutinogen preparationas provided herein. The immunogenic composition may be formulated as avaccine for in vivo use for protecting a host immunized therewith fromdisease caused by Bordetella and may comprise at least one otherBordetella antigen. The at least one other Bordetella antigen may befilamentous haemagglutinin, the 69 kDa outer membrane protein adenylatecyclase, Bordetella lipooligosaccharide, outer membrane proteins andpertussis toxin or a toxoid thereof, including genetically detoxifiedanalogs thereof.

[0018] In a further aspect of the invention, the immunogenic compositionas provided herein may comprise at least one non-Bordetella immunogen.Such non-Bordetella immunogen may be diphtheria toxoid, tetanus toxoid,fcapsular pooysaccharide of Haemophilus, outer membrane protein ofHaemoophilus, hepatitis B surface antigen, polio, mumps, measles and/orrubella.

[0019] The immunogenic compositions as provided herein may furthercomprise an adjuvant and such adjuvant may be aluminum phosphate,aluminum hydroxide, Quil A, QS21, calcium phosphate, calcium hydroxide,zinc hydroxide, a glycolipid analog, an octodecyl ester of an amino acidor a lipoprotein.

[0020] In accordance with one aspect of the present invention, there isprovided a vaccine composition for protecting an at-risk humanpopulation against a case of disease caused by infection by B.pertussis, which comprises pertussis toxoid, filamentous haemagglutinin,pertactin and agglutinogens in purified form in selected relativeamounts to confer protection to the extent of at least about 70% ofmembers of the at-risk population.

[0021] Such vaccine composition may contain about 5 to about 30 μgnitrogen of pertussis toxoid, about 5 to about 30 μg nitrogen offilamentous haemagglutinin, about 3 to about 15 μg nitrogen of pertactinand about 1 to about 10 μg nitrogen of agglutinogens.

[0022] In one specific embodiment, the vaccine may comprise pertussistoxoid, filamentous haemagglutinin, the 69 kDa protein and filamentousagglutinogens of Bordetella at a weight ratio of about 10:5:5:3 asprovided by about 10 μg of pertussis toxoid, about 5 μg of filamentoushaemagglutinin, about 5 μg of 69 kDa protein and about 3 μg of fimbrialagglutinogens in a single human dose. In a further particularembodiment, the vaccine may comprise pertussis toxoid, filamentoushaemagglutinin, 69 kDa protein and fimbrial agglutinogens in a weightratio of about 20:20:5:3 as provided by about 20 μg of pertussis toxoid,about 20 μg of filamentous haemagglutinin, about 5 μg of 69 kDa proteinand about 3 μg of fimbrial agglutinogens in a single human dose. In ayet further particular embodiment, the vaccine may comprise pertussistoxoid filamentous haemagglutinin, 69 kDa protein and fimbrialagglutinoaens in a weight ratio of about 20:10:10:6 as provided by about20 μg of pertussis toxoid, about 10 μg of filamentous haemagglutinin,about 10 μg of 69 kDa protein and about 6 μg of fimbrial agglutinogensin a single human dose.

[0023] The extent of protection to the at-risk human population affordedby the vaccine composition of the invention may be at least about 80%,preferably about 85%, for a case of spasmodic cough of duration at least21 days and culture-confirmed bacterial infection. The extent ofprotection to the at-risk human population may be at least about 70% fora case of mild pertussis having a cough of at least one day duration.

[0024] The agglutinogens component of the vaccine preferably comprisefimbrial agglutinogen 2 (Agg 2) and fimbrial agglutinogen 3 (Agg 3)substantially free from agglutinogen 1. The weight ratio of Agg 2 to Agg3 may be from about 1.5:1 to about 2:1.

[0025] The vaccine provided herein may be combined with tetanus toxoidand diphtheria toxoid to provide a DTP vaccine. In one embodiment, thevaccine contains about 15 Lfs of diphtheria toxoid and about 5 Lfs oftetanus toxoid.

[0026] In addition, the vaccine may also comprise an adjuvant,particularly alum.

[0027] In a further aspect of the present invention, there is provided amethod of immunizing an at-risk human population against disease causedby infection by B. pertussis, which comprises administering to membersof the at-risk human population an immunoeffective amount of the vaccinecomposition provided herein to confer protection to the extent of atleast about 70% of the members of the at-risk population.

[0028] Advantages of the present invention include an improved acellularpertussis vaccine composition of increased efficacy.

[0029] The present invention further provides, in an additional aspect,purified forms of pertussis toxin, filamentous haemagglutinin, pertactinand fimbrial agglutinogens of B. pertussis when used in the manufactureof a vaccine composition for administration to an at-risk humanpopulation to confer protection to the extent of at least about 70% ofthe members of said at-risk human population.

[0030] In such use, there may be employed in the manufacture of a singlehuman dose of the vaccine composition from about 30 μg of nitrogen ofpertactin and about 1 to about 10 μg of nitrogen of the fimbrialagglutinogens. In particular, the vaccine composition as provided hereinhave been selected by the National Institute of Allergy and InfectiousDiseases (NIAID) of the United States Government for evaluation in adouble-blind, human efficacy clinical trial, thereby establishing asufficient basis to those especially skilled in the art that thecompositions will be effective to some degree in preventing the stateddisease (pertussis). The subject of that trial (being a vaccine asprovided herein) has met the burden of being reasonably predictive ofutility.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The present invention will be further understood from thefollowing detailed description and Examples with reference to theaccompanying drawing in which:

[0032]FIG. 1 is a schematic flow sheet of a procedure for the isolationof an agglutinogen preparation from a Bordetella strain.

DETAILED DESCRIPTION OF THE INVENTION

[0033] Referring to FIG. 1, there is illustrated a flow sheet of amethod for preparing an agglutinogen preparation from a Bordetellastrain. As seen in FIG. 1, a Bordetella cell paste containing theagglutinogens, such as B. pertussis cell paste, is extracted with, forexample, a urea-containing buffer, such as 10 mM potassium phosphate,150 mM NaCl and 4M urea, to selectively extract the agglutinogens fromthe cell paste to produce a first supernatant (sp1) containingagglutinogens and a first residual precipitate (ppt1). The firstsupernatant (sp1) is separated from the first residual precipitate(ppt1) such as by centrifugation. The residual precipitate (ppt1) isdiscarded. The clarified supernatant (sp1) then may be concentrated anddiafiltered against, for example, 10 mM potassium phosphate/150 mMNaCl/0.1% Triton X-100 using, for example, a 100 to 300 kDa NMWLmembrane filter.

[0034] The first supernatant then is incubated at a temperature and fora time to produce a clarified supernatant (sp2) containing agglutinogensand a second discard precipitate (ppt2) containing non-agglutinogencontaminants. Appropriate temperatures include about 50° C. to about100° C., including about 75° to about 85° C., and appropriate incubationtimes include about 1 to about 60 minutes. The clarified supernatantthen is concentrated by, for example, the addition of polyethyleneglycol of molecular weight about 8000 (PEG 8000) to a finalconcentration of about 4.5±0.2% and stirring gently for a minimum ofabout 30 minutes to produce a third precipitate (ppt3) which may becollected by centrifugation. The remaining supernatant sp3 is discarded.

[0035] This third precipitate (ppt3) is extracted with, for example, abuffer comprising 10 mM potassium phosphate/150 mM NaCl to provide thecrude fimbrial agglutinogen-containing solution. 1M potassium phosphatemay be added to the crude fimbrial solution to make it about 100 mM withrespect to potassium phosphate. Alternatively, the clarified supernatantof heat-treated fimbrial agglutinogens can be purified withoutprecipitation by gel-filtration chromatography using a gel, such asSepharose CL6B. The fimbrial agglutinogens in the crude solution thenare purified by column chromatography, such as, by passing through a PEIsilica column, to produce the fimbrial agglutinogen preparation in therun-through.

[0036] This fimbrial agglutinogen containing run-through may be furtherconcentrated and diafiltered against, for example, a buffer containing10 mM potassium phosphate/150 mM NaCl using a 100-300 kDa NMWL membrane.The agglutinogen preparation may be sterilized by filtration through a≦0.22 μM membrane filter, to provide the final purified fimbrialagglutinogen preparation containing fimbrial agglutinogen 2 and 3.

[0037] An agglutinogen preparation from a Bordetella strain may comprisefimbrial agglutinogen 2 (Agg 2) and fimbrial agglutinogen 3 (Agg 3)substantially free from agglutinogen 1. The weight ratio of Agg 2 to Agg3 may be from about 1.5:1 to about 2:1. Such fimbrial agglutinogenpreparations may be produced by the method as provided herein anddescribed in detail above. The present invention also extends toimmunogenic compositions (including vaccines) comprising the fimbrialagglutinogen preparations provided as described above. Such vaccinescontain other Bordetella immunogens, including filamentoushaemagglutinin, the 69 kDa outer membrane protein and pertussis toxin ora toxoid thereof, including genetically detoxified analogs of PT asdescribed in, for example, ref. 68.

[0038] Such vaccines may include non-Bordetella immunogens includingdiphtheria toxoid, tetanus toxoid, capsular polysaccharide ofHaemophilus, outer membrane protein of Haemophilus, hepatitis B surfaceantigen, polio, mumps, measles and rubella.

[0039] Each of the Bordetella antigens is individually absorbed toadjuvant (such as alum) to provide for convenient and rapid productionof vaccines containing selected relative amounts of antigens in vaccinesas provided herein in order to confer protection to an extent of atleast about 70% of the members of an at risk population, preferably atleast about 80% of such population.

[0040] In selected embodiments, the invention provides vaccines with thefollowing characteristics (Ag proteins used herein are based on Kjedahltest results performed on purified concentrates and are expressed as /gof protein nitrogen), all of which may be administered by intramuscularinjection:

[0041] (a) CP_(10/5/5/3)DT

[0042] One formulation of component pertussis vaccine combined withdiphtheria and tetanus toxoids was termed CP_(10/5/5/3)DT. Each 0.5 mlhuman dose of CP_(10/5/5/3)DT was formulated to contain about: 10 μgPertussis toxoid (PT)  5 μg Filamentous haemagglutonin (FHA)  5 μgFimbrial agglutinogens 2 and 3 (FIMB)  3 μg 69 kDa outer membraneprotein 15 Lf Diphtheria toxoid  5 Lf Tetanus toxoid  1.5 mg Aluminumphosphate  0.6% 2-phenoxyethanol, as preservative

[0043] (b) CP_(20/20/5/3)DT

[0044] Another formulation of component pertussis vaccine combined withdiphtheria and tetanus toxoids was termed CP_(20/20/5/3)DT. Each 0.5 mlhuman dose of CP_(20/20/5/3)DT was formulated to contain about: 20 μgPertussis toxoid (PT) 20 μg Filamentous haemagglutonin (FHA)  5 μgFimbrial agglutinogens 2 and 3 (FIMB)  3 μg 69 kDa outer membraneprotein 15 Lf Diphtheria toxoid  5 Lf Tetanus toxoid  1.5 mg Aluminumphosphate  0.6% 2-phenoxyethanol, as preservative

[0045] (c) CP_(10//5/5)DT

[0046] One formulation of component pertussis vaccine combined withdiphtheria and tetanus toxoids was termed CP_(10/5/5)DT. Each 0.5 mLhuman dose of CP_(10/5/5) was formulated to contain about: 10 μgPertussis toxoid (PT)  5 μg Filamentous haemagglutonin (FHA)  5 μgFimbrial agglutinogens 2 and 3 (FIMB) 15 Lf Diphtheria toxoid  5 LfTetanus toxoid  1.5 mg Aluminum phosphate  0.6% 2-phenoxyethanol aspreservative

[0047] (d) CP_(20/10/10/6)DT

[0048] A further formulation of component pertussis vaccine combinedwith diphtheria and tetanus toxoids was termed CP_(20/10/10/6)DT. Each0.5 ml human dose of CP_(20/10/10/6)DT was formulated to contain about:20 μg Pertussis toxoid (PT) 10 μg Filamentous haemagglutonin (FHA) 10 μgFimbrial agglutinogens 2 and 3 (FIMB)  6 μg 69 kDa outer membraneprotein (69 kDA) 15 Lf Diphtheria toxoid  5 Lf Tetanus toxoid  1.5 mgAluminum phosphate  0.6% 2-phenoxyethanol, as preservative

[0049] The other Bordetella immunogens, pertussis toxin (includinggenetically detoxified analogs thereof, as described in, for example,Klein et al, U.S. Pat. No. 5,085,862 assigned to the assignee hereof andincorporated herein by reference thereto), FHA and the 69 kDa proteinmay be produced by a variety of methods such as described below:

Purification of PT

[0050] PT may be isolated from the culture supernatant of a B. pertussisstrain using conventional methods. For example, the method of Sekura etal (ref. 55) may be used. PT is isolated by first absorbing culturesupernatant onto a column containing the dye-ligand gel matrix, Affi-GelBlue (Bio-Rad Laboratories, Richmond, Calif.). PT is eluted from thiscolumn by high salt, such as, 0.75 M magnesium chloride and, afterremoving the salt, is passed through a column of fetuin-Sepharoseaffinity matrix composed of fetuin linked to cyanogen bromide-activatedSepharose. PT is eluted from the fetuin column using 4M magnesium salt.

[0051] Alternatively, the method of Irons et al (ref. 56) may be used.Culture supernatant is absorbed onto a CNBr-activated Sepharose 4Bcolumn to which haptoglobin is first covalently bound. The PT binds tothe absorbent at pH 6.5 and is eluted from the column using 0.1MTris/0.5M NaCl buffer by a stepwise change to pH 10.

[0052] Alternatively, the method described in U.S. Pat. No. 4,705,686granted to Scott et al on Nov. 10, 1987 and incorporated herein byreference thereto may be used. In this method culture supernatants orcellular extracts of B. pertussis are passed through a column of ananion exchange resin of sufficient capacity to adsorb endotoxin butpermit Bordetella antigens to flow through or otherwise be separatedfrom the endotoxin.

[0053] Alternatively, PT may be purified by using perlitechromatography, as described in EP Patent No. 336 736, assigned to theassignee thereof and incorporated herein by reference thereto.

Detoxification of PT

[0054] PT is detoxified to remove undesired activities which could causeside reactions of the final vaccine. Any of a variety of conventionalchemical detoxification methods can be used, such as treatment withformaldehyde, hydrogen peroxide, tezranitro-methane, or glutaraldehyde.

[0055] For example, PT can be detoxified with glutaraldehyde using amodification of the procedure described in Munoz et al (ref. 57). Inthis detoxification process purified PT is incubated in a solutioncontaining 0.01 M phosphate buffered saline. The solution is made 0.05%with glutaraldehyde and the mixture is incubated at room temperature fortwo hours, and then made 0.02 M with L-lysine. The mixture is furtherincubated for two hours at room temperature and then dialyzed for twodays against 0.01 M PBS. In a particular embodiment, the detoxificationprocess of EP Patent No. 336 736 may be used. Briefly PT may bedetoxified with glutaraldehyde as follows:

[0056] Purified PT in 75 mM potassium phosphate at pH 8.0 containing0.22M sodium chloride is diluted with an equal volume of glycerol toprotein concentrations of approximately 50 to 400 μg/ml. The solution isheated to 37° C. and detoxified by the addition of glutaraldehyde to afinal concentration of 0.5% (w/v). The mixture is kept at 37° C. for 4hrs and then aspartic acid (1.5 M) is added to a final concentration of0.25 M. The mixture is incubated at room temperature for 1 hour and thendiafiltered with 10 volumes of 10 mM potassium phosphate at pH 8.0containing 0.15M sodium chloride and 5% glycerol to reduce the glyceroland to remove the glutaraldehyde. The PT toxoid is sterile-filteredthrough a 0.2 μM membrane.

[0057] If recombinant techniques are used to prepare a PT mutantmolecule which shows no or little toxicity, for use as the toxoidedmolecule, chemical detoxification is not necessary.

Purification of FHA

[0058] FHA may be purified from the culture supernatant essentially asdescribed by Cowell et al (ref. 58). Growth promoters, such asmethylated beta-cyclodextrins, may be used to increase the yield of FHAin culture supernatants. The culture supernatant is applied to ahydroxylapatite column. FHA is adsorbed onto the column, but PT is not.The column is extensively washed with Triton X-100 to remove endotoxin.FHA is then eluted using 0.5M NaCl in 0.1M sodium phosphate and, ifneeded, passed through a fetuin-Sepharose column to remove residual PT.Additional purification can involve passage though a Sepharose CL-6Bcolumn.

[0059] Alternatively, FHA may be purified using monoclonal antibodies tothe antigen, where the antibodies are affixed to a CNBr-activatedaffinity column (ref. 59).

[0060] Alternatively, FHA may be purified by using perlitechromatography as described in the above-mentioned EP 336 736.

Purification of 69 kDa Outer Membrane Protein (pertactin)

[0061] The 69 kDa outer membrane protein (69K or pertactin) may berecovered from bacterial cells by first inactivating the cells with abacteriostatic agent, such as thimerosal, as described in published EP484 621 and incorporated herein by reference thereto. The inactivatedcells are suspended in an aqueous medium, such as PBS (pH 7 to 8) andsubjected to repeated extraction at elevated temperature (45 to 60° C.)with subsequent cooling to room temperature or 4° C. The extractionsrelease the 69K protein from the cells. The material containing the 69Kprotein is collected by precipitation and passed through an Affi-gelBlue column. The 69K protein is eluted with a high concentration ofsalt, such as G.5M magnesium chloride. After dialysis, it is passedthrough a chromatofocusing support.

[0062] Alternatively, the 69 kDa protein may be purified from theculture supernatant of a B. pertussis culture, as described in publishedPOT Application WO 91/15505, in the name of the assignee hereof andincorporated herein by reference thereto.

[0063] Other appropriate methods of purification of the 69 kDa outermembrane protein from B. pertussis are described in U.S. Pat. No.5,276,142, granted to Gotto et al on Jan. 4, 1984 and in U.S. Pat. No.5,101,014, granted to Burns on Mar. 31, 1992.

[0064] A number of clinical trials were performed in humans as describedherein to establish the safety, non-reactogenicity and utility ofcomponent vaccines for protection against pertussis. In particular,immune responses to each of the antigens contained in the vaccines (asshown, for example, in Table 3 below) were obtained. One particularacellular pertussis vaccine CP_(10/5/5/3)DT was analyzed in a largeplacebo-controlled, multi-centre, double-randomized clinical trial in anat-risk human population to estimate the efficacy of the vaccine againsttypical pertussis.

[0065] The case definition for typical pertussis disease was:

[0066] Twenty-one days or more of spasmodic cough, and either

[0067] culture-confirmed B. pertussis, or

[0068] serological evidence of Bordetella specific infection indicatedby a 100% IgG or IgA antibody rise in ELISA against FHA or PT in pairedsera, or

[0069] if serological data is lacking, the study child has been incontact with a case of culture-confirmed B. pertussis in the householdwith onset of cough within 28 days before or after the onset of cough inthe study child.

[0070] The results of this study showed CP_(10/5/5/3)DT to be about 85%efficacious in preventing pertussis as defined in the case definitionfor typical pertussis disease as described above. In the same study, atwo-component pertussis acellular vaccine containing only PT and FHA wasabout 58% efficacious and a whole-cell pertussis vaccine was about 48%efficacious (see Table 4 below). In addition, the CP_(10/5/5/3)DTvaccine prevented mild pertussis defined as a cough of at least one dayduration to an efficacy of about 77%. In particular, the profile ofimmune response obtained was substantially the same as that obtainedfollowing immunization with whole-cell pertussis vaccines which arereported to be highly efficacious against pertussis.

Vaccine Preparation and Use

[0071] Thus, immunogenic compositions, suitable to be used as vaccines,may be prepared from the Rordetella immunogens as disclosed herein. Thevaccine elicits an immune response in a subject which producesantibodies that may be opsonizing or bactericidal. Should the vaccinatedsubject be challenged by B. pertussis, such antibodies bind to andinactivate the bacteria. Furthermore, opsonizing or bactericidalantibodies may also provide protection by alternative mechanisms.

[0072] Immunogenic compositions including vaccines may be prepared asinjectibles, as liquid solutions or emulsions. The Bordetella immunogensmay be mixed with pharmaceutically acceptable excipients which arecompatible with the immunogens. Such excipients may include water,saline, dextrose, glycerol, ethanol, and combinations thereof. Theimmunogenic compositions and vaccines may further contain auxiliarysubstances, such as wetting or emulsifying agents, pH buffering agents,or adjuvants to enhance the effectiveness thereof. Immunogeniccompositions and vaccines may be administered parenterally, by injectionsubcutaneously or intramuscularly. The immunogenic preparations andvaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective,immunogenic and protective. The quantity to be administered depends onthe subject to be treated, including, for example, the capacity of theimmune system of the individual to synthesize antibodies, and, ifneeded, to produce a cell-mediated immune response. Precise amounts ofactive ingredient required to be administered depend on the judgment ofthe practitioner. However, suitable dosage ranges are readilydeterminable by one skilled in the art and may be of the order ofmicrograms of the immunogens. Suitable regimes for initialadministration and booster doses are also variable, but may include aninitial administration followed by subsequent administrations. Thedosage may also depend on the route of administration and will varyaccording to the size of the host.

[0073] The concentration of the immunogens in an immunogenic compositionaccording to the invention is in general about 1 to about 95%. A vaccinewhich contains antigenic material of only one pathogen is a monovalentvaccine. Vaccines which contain antigenic material of several pathogensare combined vaccines and also belong to the present invention. Suchcombined vaccines contain, for example, material from various pathogensor from various strains of the same pathogen, or from combinations ofvarious pathogens.

[0074] Immunogenicity can be significantly improved if the antigens areco-administered with adjuvants, commonly used as 0.005 to 0.5 percentsolution in phosphate buffered saline. Adjuvants enhance theimmunogenicity of an antigen but are not necessarily immunogenicthemselves. Adjuvants may act by retaining the antigen locally near thesite of administration to produce a depot effect facilitating a slow,sustained release of antigen to cells of the immune system. Adjuvantscan also attract cells of the immune system to an antigen depot andstimulate such cells to elicit immune responses.

[0075] Immunostimulatory agents or adjuvants have been used for manyyears to improve the host immune responses to, for example, vaccines.Intrinsic adjuvants, such as lipopolysaccharides, normally are thecomponents of the killed or attenuated bacteria used as vaccines.Extrinsic adjuvants are immunomodulators which are typicallynon-covalently linked to antigens and are formulated to enhance the hostimmune responses. Thus, adjuvants have been identified that enhance theimmune response to antigens delivered parenterally. Some of theseadjuvants are toxic, however, and can cause undesirable side-effects,making them unsuitable for use in humans and many animals. Indeed, onlyaluminum hydroxide and aluminum phosphate (collectively commonlyreferred to as alum) are routinely used as adjuvants in human andveterinary vaccines. The efficacy of alum in increasing antibodyresponses to diphtheria and tetanus toxoids is well established and,more recently, a HBsAg vaccine has been adjuvanted with alum. While theusefulness of alum is well established for some applications, it haslimitations. For example, alum is ineffective for influenza vaccinationand inconsistently elicits a cell mediated immune response. Theantibodies elicited by alum-adjuvanted antigens are mainly of the IgGlisotype in the mouse, which may not be optimal for protection by somevaccinal agents.

[0076] A wide range of extrinsic adjuvants can provoke potent immuneresponses to antigens. These include saponins complexed to membraneprotein antigens (immune stimulating complexes), pluronic polymers withmineral oil, killed mycobacteria in mineral oil, Freund's completeadjuvant, bacterial products, such as muramyl dipeptide (MDP) andlipopolysaccharide (LPS), as well as lipid A, and liposomes.

[0077] To efficiently induce humoral immune responses (HIR) andcell-mediated immunity (CMI), immunogens are often emulsified inadjuvants. Many adjuvants are toxic, inducing granulomas, acute andchronic inflammations (Freund's complete adjuvant, FCA), cytolysis(saponins and Pluronic polymers) and pyrogenicity, arthritis andanterior uveitis (LPS and MDP) . Although FCA is an excellent adjuvantand widely used in research, it is not licensed for use in human orveterinary vaccines because of its toxicity.

[0078] Desirable characteristics of ideal adjuvants include:

[0079] (1) lack of toxicity;

[0080] (2) ability to stimulate a long-lasting immune response;

[0081] (3) simplicity of manufacture and stability in long-term storage;

[0082] (4) ability to elicit both CMI and HIR to antigens administeredby various routes;

[0083] (5) synergy with other adjuvants;

[0084] (6) capability of selectively interacting with populations ofantigen presenting cells (APC):

[0085] (7) ability to specifically elicit appropriate T_(H) ¹ or T_(H) ²cell-specific immune responses; and

[0086] (8) ability to selectively increase appropriate antibody isotypelevels (for example, IgA) against antigens.

[0087] U.S. Pat. No. 4,855,283 granted to Lockhoff et al on Aug. 8, 1989which is incorporated herein by reference thereto teaches glycolipidanalogues including N-glycosylamides, N-glycosylureas andN-glycosylcarbamates, each of which is substituted in the sugar residueby an amino acid, as immuno-modulators or adjuvants. Thus, Lockhoff etal. (U.S. Pat. No. 4,855,283 and ref. 60) reported that N-glycolipidanalogs displaying structural similarities to the naturally-occurringglycolipids, such as glycosphingolipids and glycoglycerolipids, arecapable of eliciting strong immune responses in both herpes simplexvirus vaccine and pseudorabies virus vaccine. Some glycolipids have beensynthesized from long chain alkylamines and fatty acids that are linkeddirectly with the sugars through the anomeric carbon atom, to mimic thefunctions of the naturally occurring lipid residues.

[0088] U.S. Pat. No. 4,258,029 granted to Moloney, assigned to theassignee hereof and incorporated herein by reference thereto, teachesthat octadecyl tyrosine hydrochloride (OTH) functions as an adjuvantwhen complexed with tetanus toxoid and formalin inactivated type I, IIand III poliomyelitis virus vaccine. Also, Nixon-George et al. (ref.61), reported that octodecyl esters of aromatic amino acids complexedwith a recombinant hepatitis B surface antigen, enhanced the host immuneresponses against hepatitis B virus.

EXAMPLES

[0089] The above disclosure generally describes the present invention. Amore complete understanding can be obtained by reference to thefollowing specific Examples. These Examples are described solely for thepurposes of illustration and are not intended to limit the scope of theinvention. Changes in form and substitution of equivalents arecontemplated as circumstances may suggest or render expedient. Althoughspecific terms have been employed herein, such terms are intended in adescriptive sense and not for purposes of limitation.

[0090] Methods of protein biochemistry, fermentation and immunology usedbut not explicitly described in this disclosure and these Examples areamply reported in the scientific literature and are well within theability of those skilled in the art.

Example 1

[0091] This Example describes the growth of Bordetella pertussis.

Master Seed

[0092] Master seed cultures of a Bordetella pertussis strain were heldas freeze-dried seed lots, at 2° C. to 8° C.

Working Seed

[0093] The freeze-dried culture was recovered in Hornibrook medium andused to seed Bordet-Gengou Agar (BGA) plates. Hornibrook medium has thefollowing composition: Component for 1 liter Casein hydrolysate(charcoal treated) 10.0 g Nicotinic acid 0.001 g Calcium chloride 0.002g Sodium chloride 5.0 g Magnesium chloride hexahydrate 0.025 g Potassiumchloride 0.200 g Potassium phosphate dibasic 0.250 g Starch 1.0 gDistilled water to 1.0 liter

[0094] The pH is adjusted to 6.9±0.1 with lo sodium carbonate solution.The medium is dispensed into tubes and sterilized by steaming in theautoclave for 20 minutes and autoclaving for 20 minutes at 121° C. to124° C. The seed was subcultured twice, firstly on BGA plates then onComponent Pertussis Agar (CPA). Component Pertussis Agar (CPA) has thefollowing composition: NaCl  2.5 g/L KH₂PO₄  0.5 g/L KCl  0.2 g/LMgCl₂(H₂O)₆  0.1 g/L Tris base  1.5 g/L Casamino acids 10.0 g/LNaHGlutamate 10.0 g/L Conc. HCl to pH 7.2 Agar 15.0 g/L Growth factors(CPGF) 10.0 mL/L

[0095] Component Pertussis Growth Factors (CPGF)—100× have the followingcomposition: L-cysteine HCl 4.0 g/L Niacin 0.4 g/L Ascorbic acid 40.0g/L Glutathione, reduced 15.0 g/L Fe₂SO₄, (H₂O)₇ 1.0 g/LDimethyl-β-cyclodextrin 100 g/L CaCl₂(H₂O)₂ 2.0 g/L

[0096] The final culture was suspended in Pertussis Seed SuspensionBuffer (CPSB), dispensed into 2 to 4 ml aliquots and stored frozen at−60° C. to −85° C. Pertussis Seed Suspension Buffer (PSSB) has thefollowing composition: Casamino acids  10.0 g/L Tris base  1.5 g/LAnhydrous glycerol 100 mL/L Conc. HCl to pH 7.2

[0097] These glycerol suspensions provided the starting material for thepreparation of the working seed.

Cultivation Process

[0098] Propagation of the working seed was conducted in ComponentPertussis Agar Roux bottles for 4 to 7 days at 34° C. to 38° C.Following this cultivation, cells were washed off agar with ComponentPertussis Broth (CPB). Samples were observed by Gram stain, for culturepurity and opacity.

[0099] Cells were transferred to 4 liter conical flasks containing CPBand incubated at 34° C. to 38° C. for 20 to 26 hours with shaking.Samples were observed by Gram stain and culture purity was checked.Flasks were pooled and the suspension was used to seed two fermenterscontaining CPB (10 liter volume starting at OD₆₀₀ 0.1-0.4). The seed wasgrown to a final OD₆₀₀ of 5.0 to 10.0. Samples were tested by Gramstrain, for culture purity, by antigen specific ELISAs and forsterility.

Example 2

[0100] This Example describes the purification of antigens from theBordetella pertussis cell culture.

Production of Broth and Cell Concentrates

[0101] Bacterial suspension was grown in two production fermenters, at34° C. to 37°°C. for 35 to 50 hours. The fermenters were sampled formedia sterility testing. The suspension was fed to a continuous-flowdisk-stack centrifuge (12,000×g) to separate cells from the broth. Cellswere collected to await extraction of fimbriae component. The clarifiedliquor was passed through ≦ 0.22 μm membrane filter. The filtered liquorwas concentrated by ultra filtration using a 10 to 30 kDa nominalmolecular weight limit (NMWL) membrane. The concentrate was stored toawait separation and purification of the Pertussis Toxin (PT),Filamentous haemagglutonin (FHA) and 69 kDa (pertactin) components.

Separation of the Broth Components

[0102] The broth components (69 kDa, PT and FHA) were separated andpurified by perlite chromatography and selective elution steps,essentially as described in EP Patent No. 336 736 and applicantspublished PCT Application No. WO 91/15505, described above. The specificpurification operations effected are described below.

Pertussis Toxin (PT)

[0103] The perlite column was washed with 50 mM Tris, 50 mM Tris/0.5%Triton X-100 and 50 mM Tris buffers. The PT fraction was eluted from theperlite column with 50 mM Tris/0.12M NaCl buffer.

[0104] The PT fraction from the perlite chromatography was loaded onto ahydroxyLapatite column and then washed with 30 mM potassium phosphatebuffer. PT was eluted with 75 mM potassium phosphate/225 mM NaCl buffer.The column was washed with 200 mM potassium phosphate/0.6M NaCl toobtain the FHA fraction which was discarded. Glycerol was added to thepurified PT to 50% and the mixture was stored at 2° C. to 8° C. untildetoxification, within one week.

Filamentous Haemagglutonin (FRA)

[0105] The FHA fraction was eluted from the perlite column with 50 mMTris/0.6M NaCl. Filamentous haemagglutinin was purified bychromatography over hydroxylapatite. The FHA fraction from the perlitecolumn was loaded onto a hydroxylapatite column then washed with 30 mMpotassium phosphate containing 0.56 Triton X-100, followed by 30 mMpotassium phosphate buffer. The PT fraction was eluted with 85 mMpotassium phosphate buffer and discarded. The FHA fraction was theneluted with 200 mM potassium phosphate/0.6M NaCl and stored at 2° C. to8° C. until detoxification within one week.

69 kDa (pertactin)

[0106] The broth concentrate was diluted with water for injection (WFI)to achieve a conductivity of 3 to 4 mS/cm and loaded onto a perlitecolumn at a loading of 0.5 to 3.5 mg protein per ml perlite. Therun-through (69 kDa Component Fraction) was concentrated byultrafiltration using a 10 to 30 kDa NMWL membrane. Ammonium sulphatewas added to the run-through concentrate to 35% ±3% (w/v) and theresulting mixture stored at 2° C. to 8° C. for 4±2 days or centrifuged(7,000×g) immediately. Excess supernatant was decanted and theprecipitate collected by centrifugation (7,000×g). The 69 kDa pellet waseither stored frozen at −20° C. to −30° C. or dissolved in Tris orphosphate buffer and used immediately.

[0107] The 69 kDa outer membrane protein obtained by the 35% (w/v)ammonium sulphate precipitation of concentrated perlite run-through wasused for the purification. Ammonium sulphate (100±5 g per liter) wasadded to the 69 kDa fraction and the mixture stirred for at least 2hours at 2° C. to 8° C. The mixture was centrifuged (7,000×g) to recoverthe supernatant. Ammonium sulphate (100 to 150 g per liter) was added tothe supernatant and the mixture stirred for at least 2 hours at 2° C. to8° C. The mixture was centrifuged (7,000×g) to recover the pellet, whichwas dissolved in 10 mM Tris, HCl, pH 8. The ionic strength of thesolution was adjusted to the equivalent of 10 mM Tris HCl (pH 8),containing 15 mM ammonium sulphate.

[0108] The 69 kDa protein was applied to a hydroxylapatite columnconnected in tandem with a Q-Sepharose column. The 69 kDa protein wascollected in the run-through, was flushed from the columns with 10 mMTris, HCl (pH 8), containing 15 mM ammonium sulphate and pooled with 69kDa protein in the run-through. The 69 kDa protein pool was diafilteredwith 6 to 10 volumes of 10 mM potassium phosphate (pH 8), containing0.15M NaCl on a 100 to 300 kDa NMWL membrane. The ultra filtrate wascollected and the 69 kDa protein in the ultra filtrate concentrated.

[0109] The 69 kDa protein was solvent exchanged into 10 mM Tris HCl(pHB), and adsorbed onto Q-Sepharose, washed with 10 mM Tris HCl (pH8)/5 mM ammonium sulphate. The 69 kDa protein was eluted with 50 mMpotassium phosphate (pH 8). The 69 kDa protein was diafiltered with 6 to10 volumes of 10 mM potassium phosphate (pH 8) containing 0.15M NaCl ona 10 to 30 kDa NMWL membrane. The 69 kDa protein was sterile filteredthrough a ≦0.22 μm filter. This sterile bulk was stored at 2° C. to 8°C. and adsorption was performed within three months.

Fimbrial Agglutinogens

[0110] The agglutinogens were purified from the cell paste followingseparation from the broth. The cell paste was diluted to a 0.05 volumefraction of cells in a buffer containing 10 mM potassium phosphate, 150mM NaCl and 4M urea and was mixed for 30 minutes. The cell lysate wasclarified by centrifugation (12,000×g) then concentrated and diafilteredagainst 10 mM potassium phosphate/150 mM NaCl/0.1% Triton X-100 using a100 to 300 kDa NMWL membrane filter.

[0111] The concentrate was heat treated at 80° C. for 30 min thenreclarified by centrifugation (9,000×g). PEG 8000 was added to theclarified supernatant to a final concentration of 4.5% ±0.2% and stirredgently for a minimum of 30 minutes. The resulting precipitate wascollected by centrifugation (17,000×g) and the pellet extracted with 10mM potassium phosphate/150 mM NaCl buffer to provide a crude fimbrialagglutinogen solution. The fimbrial agglutinogens were purified bypassage over PEI silica. The crude solution was made 100 mM with respectto potassium phosphate using 1M potassium phosphate buffer and passedthrough the PEI silica column.

[0112] The run-through from the columns was concentrated and diafilteredagainst 10 mM potassium phosphate/150 mM NaCl buffer using a 100 to 300kDa NMWL membrane filter. This sterile bulk is stored at 2° C. to 8° C.and adsorption performed within three months. The fimbrial agglutinogenpreparation contained fimbrial Agg 2 and fimbrial Agg 3 in a weightratio of about 1.5 to about 2:1 and was found to be substantially freefrom Agg 1.

Example 3

[0113] This Example describes the toxoiding of the purified Bordetellapertussis antigens, PT and FHA.

[0114] PT, prepared in pure form as described in Example 2, was toxoidedby adjusting the glutaraldehyde concentration in the PT solution to 0.5%±0.1% and incubating at 37° C.±3° C. for 4 hours. The reaction wasstopped by adding L-aspartate to 0.21±0.02M. The mixture was then heldat room temperature for 1±0.1 hours and then at 2° C. to 8° C. for 1 to7 days.

[0115] The resulting mixture was diafiltered against 10 mM potassiumphosphate/0.15M NaCl/5% glycerol buffer on a 30 kDa NMWL membrane filterand then sterilized by passage through a ≦0.22 μm membrane filter. Thissterile bulk was stored at 2° C. to 8° C. and adsorption performedwithin three months.

[0116] The FHA fraction, prepared in pure form as described in Example2, was toxoided by adjusting the L-lysine and formaldehyde concentrationto 47±5 mM and 0.24±0.05% respectively and incubating at 35° C. to 38°C. for 6 weeks. The mixture was then diafiltered against 10 mM potassiumphosphate/0.5M NaCl using a 30 kDa NMWL membrane filter and sterilizedby passage through a membrane filter. This sterile bulk was stored a 2°C. to 8° C. and adsorption performed within three months.

Example 4

[0117] This Example describes the adsorption of the purified Bordetellapertussis antigens.

[0118] For the individual adsorption of PT, FHA, Agg and 69 kDa ontoaluminum phosphate (alum), a stock solution of aluminum phosphate wasprepared to a concentration of 18.75±1 mg/ml. A suitable vessel wasprepared and any one of the antigens aseptically dispensed into thevessel. 2-phenoxyethanol was aseptically added to yield a finalconcentration of 0.6% ±0.1% v/v and stirred until homogeneous. Theappropriate volume of aluminum phosphate was aseptically added into thevessel. An appropriate volume of sterile distilled water was added tobring the final concentration to 3 mg aluminum phosphate/ml. Containerswere sealed and labelled and allowed to stir at room temperature for 4days. The vessel was then stored awaiting final formulation.

Example 5

[0119] This Example describes the formulation of a component pertussisvaccine combined with diphtheria and tetanus toxoids.

[0120] The B. pertussis antigens prepared as described in the precedingExamples were formulated with diphtheria and tetanus toxoids to provideseveral component pertussis (CP) vaccines.

[0121] The pertussis components were produced from Bordetella pertussisgrown in submerged culture as described in detail in Examples 1 to 4above. After completion of growth, the culture broth and the bacterialcells were separated by centrifugation. Each antigen was purifiedindividually. Pertussis toxin (PT) and Filamentous Haemagglutinin (FHA)were purified from the broth by sequential chromatography over perliteand hydroxylapatite. PT was detoxified with glutaraldehyde and anyresidual PT (approximately 1%) present in the FHA fraction wasdetoxified with formaldehyde. Fimbrial Agglutinogens (2+3) (AGG) wereprepared from the bacterial cells. The cells were disrupted with ureaand heat treated, and the fimbrial agglutinogens were purified byprecipitation with polyethylene glycol and chromatography overpolyethyleneimine silica. The 69 kDa protein (pertactin) component wasisolated from the run through from the perlite chromatography step(Example 2) by ammonium sulphate precipitation, and purified bysequential chromatography over hydroxylapatite and Q-sepharose. Allcomponents were sterilized by filtration through a 0.22 μm membranefilter.

[0122] Diphtheria toxoid was prepared from Corynebacterium diphtheriaegrown in submerged culture by standard methods. The production ofDiphtheria Toxoid is divided into five stages, namely maintenance of theworking seed, growth of Corynebacterium diphtheriae, harvest ofDiphtheria Toxin, detoxification of Diphtheria Toxin and concentrationof Diphtheria Toxoid.

Preparation of Diphtheria Toxoid

[0123] (I) Working Seed

[0124] The strain of Corynebacterium diphtheriae was maintained as afreeze-dried seed lot. The reconstituted seed was grown on Loefflerslopes for 18 to 24 hours at 35° C.±2° C., and then transferred toflasks of diphtheria medium. The culture was then tested for purity andLf content. The remaining seed was used to inoculate a fermenter.

[0125] (II) Growth of Corynebacterium diphtheriae

[0126] The culture was incubated at 35° C.±2° C. and agitated in thefermenter. Predetermined amounts of ferrous sulphate, calcium chlorideand phosphate solutions were added to the culture. The actual amounts ofeach solution (phosphate, ferrous sulphate, calcium chloride) weredetermined experimentally for each lot of medium. The levels chosen arethose which gave the highest Lf content. At the end of the growth cycle(30 to 50 hours), the cultures were sampled for purity, and Lf content.

[0127] The pH was adjusted with sodium bicarbonate, and the cultureinactivated with 0.4% toluene for 1 hour at a maintained temperature of35° C.±2° C. A sterility test was then performed to confirm the absenceof live C. diphtheriae.

[0128] (III) Harvest of Diphtheria Toxin

[0129] The toluene treated cultures from one or several fermenters werepooled into a large tank. Approximately 0.12% sodium bicarbonate, 0.25%charcoal, and 23% ammonium sulphate were added, and the pH was tested.

[0130] The mixture was stirred for about 30 minutes. Diatomaceous earthwas added and the mixture pumped into a depth filter. The filtrate wasrecirculated until clear, then collected, and sampled for Lf contenttesting. Additional ammonium sulphate was added to the filtrate to givea concentration of 40%. Diatomaceous earth was also added. This mixturewas held for 3 to 4 days at 2° C. to 8° C. to allow the precipitate tosettle. Precipitated toxin was collected and dissolved in 0.9% saline.The diatomaceous earth was removed by filtration and the toxin dialysedagainst 0.9% saline, to remove the ammonium sulphate. Dialysed toxin waspooled and sampled for Lf content and purity testing.

[0131] (IV) Detoxification of Diphtheria Toxin

[0132] Detoxification takes place immediately following dialysis. Fordetoxification, the toxin was diluted so that the final solutioncontained:

[0133] a) diphtheria toxin at 1000±10% Lf/ml.

[0134] b) 0.5% sodium bicarbonate

[0135] c) 0.5% formalin

[0136] d) 0.9% w/v L-lysine monohydrochloride

[0137] The solution was brought up to volume with saline and the pHadjusted to 7.6±0.1.

[0138] Toxoid was filtered through cellulose diatomaceous earth filterpads and/or a membrane prefilter and 0.2 μm membrane filter into thecollection vessel and incubated for 5 to 7 weeks at 34° C. A sample waswithdrawn for toxicity testing.

[0139] (V) Concentration of Purified Toxoid

[0140] The toxoids were pooled, then concentrated by ultrafiltration,and collected into a suitable container. Samples were taken for Lfcontent and purity testing. The preservative (2-phenoxyethanol) wasadded to give a final concentration of 0.375% and the pH adjusted to 6.6to 7.6.

[0141] The toxoid was sterilized by filtration through a prefilter and a0.2 μm membrane filter (or equivalent) and collected. The sterile toxoidwas then sampled for irreversibility of toxoid Lf content, preservativecontent, purity (nitrogen content), sterility and toxicity testing. Thesterile concentrated toxoid was stored at 2° C. to 8° C. until finalformulation.

Preparation of Tetanus Toxoid

[0142] Tetanus toxoid (T) was prepared from Clostridium tetani grown insubmerged culture.

[0143] The production of Tetanus Toxoid can be divided into five stages,namely maintenance of the working seed, growth of Clostridium tetani,harvest of Tetanus Toxin, detoxification of Tetanus Toxin andpurification of Tetanus Toxoid.

[0144] (I) Working Seed

[0145] The strain of Clostridium tetani used in the production oftetanus toxin for the conversion to tetanus toxoid was maintained in thelyophilized form in a seed-lot. The seed was inoculated intothioglycollate medium and allowed to grow for approximately 24 hours at35° C.±2° C. A sample was taken for culture purity testing.

[0146] (II) Growth of Clostridium tetani

[0147] The tetanus medium was dispensed into a fermenter, heat-treatedand cooled. The fermenter was then seeded and the culture allowed togrow for 4 to 9 days at 34° C.± 2° C. A sample was taken for culturepurity, and Lf content testing.

[0148] (III) Harvest of Tetanus Toxin

[0149] The toxin was separated by filtration through cellulosediatomaceous earth pads, and the clarified toxin then filter-sterilizedusing membrane filters. Samples were taken for Lf content and sterilitytesting. The toxin was concentrated by ultrafiltration, using a poresize of 30,000 daltons.

[0150] (IV) Detoxification of Tetanus Toxin

[0151] The toxin was sampled for Lf content testing prior todetoxification. The concentrated toxin (475 to 525 Lf/ml) was detoxifiedby the addition of 0.5% w/v sodium bicarbonate, 0.3% v/v formalin and0.9% w/v L-lysine monohydrochloride and brought up to volume withsaline. The pH was adjusted to 7.5±0.1 and the mixture incubated at 37°C. for 20 to 30 days. Samples were taken for sterility and toxicitytesting.

[0152] (V) Purification of Toxoid

[0153] The concentrated toxoid was sterilized through pre-filters,followed by 0.2 μm membrane filters. Samples were taken for sterilityand Lf content testing.

[0154] The optimum concentration of ammonium sulphate was based on afractionation “S” curve determined from samples of the toxoid. The firstconcentration was added to the toxoid (diluted to 1900-2100 Lf/ml). Themixture was kept for at least 1 hour at 20° C. to 25° C. and thesupernatant collected and the precipitate containing the high molecularweight fraction, discarded.

[0155] A second concentration of ammonium sulphate was added to thesupernatant for the second fractionation to remove the low molecularweight impurities. The mixture was kept for at least 2 hours at 20° C.to 25° C. and then could be held at 2° C. to 8° C. for a maximum ofthree days. The precipitate, which represents the purified toxoid, wascollected by centrifugation and filtration.

[0156] Ammonium sulphate was removed from the purified toxoid bydiafiltration, using Amicon (or equivalent) ultrafiltration membraneswith PBS until no more ammonium sulphate could be detected in the toxoidsolution. The pH was adjusted to 6.6. to 7.6, and 2-phenoxyethanol addedto give a final concentration of 0.375%. The toxoid was sterilized bymembrane filtration, and samples are taken for testing (irreversibilityof toxoid, Lf content, pH, preservative content, purity, sterility andtoxicity).

[0157] One formulation of a component pertussis vaccine combined withdiphtheria and tetanus toxoids was termed CP_(10/5/5/3)DT, Each 0.5 mlhuman dose of CP_(10/5/5/3)DT was formulated to contain: 10 μg Pertussistoxoid (PT)  5 μg Filamentous haemagglutonin (FHA)  5 μg Fimbrialagglutinogens 2 and 3 (FIMB)  3 μg 69 kDa outer membrane protein 15 LfDiphtheria toxoid  5 Lf Tetanus toxoid  1.5 mg Aluminum phosphate  0.6%2-phenoxyethanol as preservative

[0158] Another formulation of component pertussis vaccine combined withdiphtheria and tetanus toxoids was termed CP_(10/5/5)DT. Each 0.5 mlhuman dose of CP_(10/5/5)DT was formulated to contain: 10 μg Pertussistoxoid (PT)  5 μg Filamentous haemagglutonin (FHA)  5 μg Fimbrialagglutinogens 2 and 3 (FIMB) 15 Lf Diphtheria toxoid  5 Lf Tetanustoxoid  1.5 mg Aluminum phosphate  0.6% 2-phenoxyethanol as preservative

[0159] Another formulation of Component Pertussis vaccine combined withdiphtheria and tetanus toxoids was termed CP_(20/20/5/3)DT. Each 0.5 mlhuman dose of CP_(20/20/5/3)DT was formulated to contain:  20 μgPertussis toxiod (PT)  20 μg Filamentous haemagglutonin (FHA)   5 μgFimbrial agglutinogens 2 and 3 (FIMB)   3 μg 69 kDa outer membraneprotein  15 Lf Diphtheria toxoid   5 Lf Tetanus toxoid 1.5 mg Aluminumphosphate 0.6% 2-phenoxyethanol as preservative

[0160] A further formulation of a component pertussis vaccine combinedwith diphtheria and tetanus toxoids was termed CP_(20/10/10/6)DT. Each0.5 ml human dose of CP_(20/10/10/6)DT was formulated to contain:  20 μgPertussis toxiod (PT)  10 μg Filamentous haemagglutonin (FHA)  10 μgFimbrial agglutinogens 2 and 3 (FIMB)   6 μg 69 kDa outer membraneprotein  15 Lf Diphtheria toxoid   5 Lf Tetanus toxoid 1.5 mg Aluminumphosphate 0.6% 2-phenoxyethanol as preservative

Example 6

[0161] This Example describes the clinical assessment of ComponentAcellular Pertussis vaccines, produced in accordance with the invention.

[0162] (a) Studies in Adults

[0163] Studies in adults and children aged 16 to 20 months indicated themulti-component vaccines containing fimbrial agglutinogens to be safeand immunogenic (Table 2).

[0164] A Phase I clinical study was performed in 17 and 18 month oldchildren in Calgary, Alberta with the five Component Pertussis vaccine(CP_(10/5/5/3)DT) and the adverse reaction reported. Thirty-threechildren received the vaccine and additionally 35 received the samevaccine without the 69 kDa protein component.

[0165] Local reactions were rare. Systemic adverse reactions, primarilyconsisting of irritability were present in approximately half of studyparticipants, regardless of which vaccine was given. Significantantibody rises were measured for anti-PT, anti-FHA, anti-fimbrialagglutinogens and anti-69 kDa IgG antibodies by enzyme immunoassay andanti-PT antibodies in the CHO cell neutralization test. No differencesin antibody response were detected in children who received the fourcomponent (CP_(10/5/5)DT) or five component (CP_(10/5/5/3)DT) except inthe anti-69 kDa antibody. Children who received the five componentvaccine containing the 69 kDa protein had a significantly higherpost-immunization anti-69 kDa antibody level.

[0166] A dose-response study was undertaken with the 4 component vaccinein Winnipeg, Manitoba, Canada. Two component vaccine formulations wereused: CP_(10/5/5/3)DT and CP_(20/10/10/6)DT, A whole-cell DPT vaccinewas also included as a control.

[0167] This study was a double-blind study in 91, 17 to 18 month oldinfants at the time of their booster pertussis dose. BothCP_(10/5/5/3)DT and CP_(20/10/10/6)DT were well tolerated by thesechildren. No differences were demonstrated in the number of children whohad any local reaction, or systemic reactions after either of thecomponent vaccines. In contrast, significantly more children whoreceived the whole-cell vaccine had local and systemic reactions thanthose who received the CP_(20/10/10/6)DT component vaccines.

Studies in Infants

[0168] Phase II:

[0169] A study was conducted using the CP_(10/5/5/3)DT vaccine inCalgary, Alberta and British Columbia, Canada. In this study, 432infants received the component pertussis vaccine or the whole-cellcontrol vaccine DPT at 2, 4 and 6 months of age. The CP_(10/5/5/3)DTvaccine was well tolerated by these infants. Local reactions were lesscommon with the component vaccine than the whole cell vaccine after eachdose.

[0170] A significant antibody response to all antigens was demonstratedafter vaccination with the component pertussis vaccine. Recipients ofthe whole-cell vaccine had a vigorous antibody response to fimbrialagglutinogens, D and T. At seven months, 82% to 89% of component vaccinerecipients and 92% of whole cell vaccine recipients had a four-foldincrease or greater rise in antibody titer to fimbrial agglutinogens. Incontrast, antibody response to FHA was 756 to 78% in component vaccinescompared to 31% of whole-cell recipients. A four-fold increase inanti-69 kDa antibody was seen in 90% to 93% of component vaccines and75% of whole-cell recipients. A four-fold rise in antibody against PT byenzyme immunoassay was seen in 40% to 49% of component vaccines and 32%of whole-cell vaccines; a four-fold rise in PT antibody by CHOneutralization was found in 55% to 69% of component and 6% of whole-cellvaccines. (Table 2).

[0171] Phase IIB:

[0172] The CP_(20/20/5/3)DT and CP_(10/10/5/3)DT vaccines were assessedin a randomized blinded study against a D₁₅PT control with a lowerdiphtheria content of 15 Lf compared to a 25 Lf formulation of 100infants at 2, 4 and 6 months of age. No differences in rates of adversereactions were detected between the two components formulations; bothwere significantly less reactogenic than the whole-cell control. Higherantibody titers against PT by enzyme immunoassay and CHO neutralizationand FHA were achieved in recipients of the CP_(20/20/5/3)DT vaccine withincreased antigen content. At 7 months, the anti-FHA geometric meantiter was 95.0 in CP_(20/20/5/3)DT recipients, 45.2 in CP_(10/5/5/3)DTrecipients were only 8.9 in D₁₅PT recipients. Anti-PT titers were 133.3,58.4 and 10.4 by immunoassay and 82.4, 32.7 and 4.0 by CHOneutralization respectively (Table 2).

[0173] This study demonstrated that the Component Pertussis vaccinecombined with diphtheria and tetanus toxoids adsorbed, with increasedantigen content, was safe and immunogenic in infants and that theincreased antigen content augmented the immune response to the preparedantigens (PT and FHA) without an increase in reactogenicity.

[0174] NIAID, PHASE II. U.S. Comparative Trial:

[0175] A phase II study was performed in the United States under theauspices of the National Institute of Allergy and Infectious Diseases(NIAID) as a prelude to a large scale efficacy trial of acellularpertussis vaccines. One component pertussis vaccine of the invention incombination with diphtheria and tetanus toxoids adsorbed(CP_(10/5/5/3)DT) was included in that trial along with 12 otheracellular vaccines and 2 whole-cell vaccines. Safety results werereported on 137 children immunized at 2, 4 and 6 months of age with theCP_(10/5/5/3)DT component vaccine.

[0176] As seen in previous studies, the component vaccine was found tobe safe, of low reactogenicity and to be well tolerated by vaccines.

[0177] At 7 months, anti-PT antibody, anti-FHA antibody, anti-69 kDaantibody and anti-fimbrial agglutinogens antibody were all higher thanor equivalent to levels achieved after the whole-cell vaccines (ref 71and Table 2). A double blind study was performed in which children wererandomly allocated to receive either the CP_(20/20/5/3)DT or theCP_(10/5/5/3)DT vaccine formulation. A total of 2050 infants wereenrolled in the United States and Canada; 1961 infants completed thestudy. Both vaccine formulations were safe, of low reactogenicity andimmunogenic in these infants. Immunogenicity was assessed in a subgroupof 292. An antibody rise was elicited to all antigens contained in thevaccine by both vaccine formulations. The CP_(20/20/5/3)DT formulationinduced higher antibody titers against FHA but not PT. TheCP_(10/5/5/3)DT formulation elicited higher titers against fimbriae andhigher agglutinogen titers.

[0178] A further safety and immunogenicity study was conducted inFrance. The study design was similar to the North American study,described above, except that vaccines were administered at 2, 3 and 4months of age. Local and systemic reactions were generally minor.Overall the vaccine was well accepted by the French study participantsusing this administration regime.

[0179] Placebo-controlled efficacy trial of two acellular pertussisvaccines and of a whole-cell vaccine in 10,000 infants

[0180] Following the results of the NIAID Phase II U.S. comparativetrial, a two-component and a five-component acellular vaccine wereselected for a multi-centre, controlled, double-randomizedplacebo-controlled efficacy trial. The clinical trial was performed inSweden, where there is a high incidence of pertussis. The two-componentvaccine contained glyceraldehyde and formalin inactivated PT (25 kg),formalin treated FHA (25 μg) and diphtheria toxoid 17 Lf and tetanustoxoid 10 Lf. The five-component pertussis vaccine was CP_(10/5/5/3)DT.For the trial, ten thousand infants, representing approximately one-halfthe infants of this age group in Sweden, were recruited in 14geographically defined study sites by use of birth registry.

[0181] Children born in January and February 1992 were randomized into a3-armed trial. After parental consent, two-thirds of the infantsreceived one out of the two diphtheria-tetanus-acellular pertussispreparations at two, four and six months of agen. The control groupreceived DT only. In May 1992, a U.S. Licensed commercially-availablewhole-cell DTP vaccine was introduced and children born in March throughDecember 1992 were randmized into a 4-armed trial. After parentalconsent, three-quarters of the infants received one out of three DTPpreparations at two, four and six months of age. The control groupreceived DT only.

[0182] Each vaccine was administered to about 2,500 children. Vaccineswere administered in three doses. The first dose was given at 2 monthsof age and not later than 3 months of age. Subsequent doses were givenwith 8 week intervals. Vaccines were given by intramuscular injection.

[0183] The children and their households were followed for 30 months. Ifpertussis was suspected, clinical data was collected, and laboratoryverification sought by nasal aspirates for bacteriological culture andpolymerase chain reaction (PCR) diagnosis. Acute and convalescent bloodsamples were collected for serological diagnosis.

[0184] Prior to this study, the extent of pertactin afforded bycomponent pertussis vaccines of the present invention in an at-riskhuman population (particularly neonates) was unknown. In particular, thecontribution of the various Bordetella components and their presence inpertussis vaccines in selected relative amounts to efficacy of thevaccines was not known.

[0185] The main aim of the trial was to estimate the ability ofacellular pertussis vaccines and whole-cell vaccine to protect againsttypical pertussis as compared to placebo.

[0186] A secondary end-point was to explore vaccine efficacy againstconfirmed pertussis infection of varying severity.

[0187] Vaccine efficacy is defined as the per cent reduction in theprobability of contracting pertussis among vaccine recipients relativeto unvaccinated children.

[0188] The relative risk of pertussis in two vaccine groups is expressedas the ratio of the disease probability in the two groups.

[0189] The probability of contracting pertussis, also called the attackrate, can be estimated in different ways. In the calculations of thesample size, the probability of contracting pertussis in a given studygroup is estimated by the quotient between the number of children withpertussis and the children remaining in the study group at thetermination of study follow-up.

[0190] The efficacy of the component vaccine CP_(10/5/5/3)DT in thistrial in preventing typical pertussis is shown in Table 4 and was about85%. In the same trial, a two-component pertussis acellular vaccinecontaining only PT and FHA was about 58% efficacious and a whole-cellvaccine was about 48% efficacious. The CP_(10/5/5/3)DT was alsoeffective in preventing mild pertussis at an estimated efficacy of about77%.

SUMMARY OF DISCLOSURE

[0191] In summary of this disclosure, the present invention providesnovel preparations of fimbrial agglutinogens of Bordetella pertussis andmethods for their production. The fimbrial agglutinogens can beformulated with other Bordetella and non-Bordetella antigens to producea number of multi-component pertussis vaccines. Such vaccines are safe,non-reactogenic, immunogenic and protective in humans. Modifications arepossible within the scope of this invention. TABLE 1 Acellular PertussisVaccines Toxoiding Refer- Vaccine PT Agent FHA P.69 AGG2 AGG3 enceAMVC + H₂O₂ ^(a) − − − − 62 Mass PHL^(b) + TMN^(c) − − − − 63 Institut +GI^(d) + − − − 64 Mérieux Smith-Kline + FI^(e)/GI + − − − 32 + FI/GI + +− − 32 CAMR^(f) + FI + − + + 65 Lederle/ + FI + + + − 66 TakedaConnaught + GI + − + + 32 + GI + + + + 67

[0192] TABLE 2 IgG antibody responses to pertussis antigen anddiphtheria and tetanus toxoids in adults and young children afterImmunization with placebo or acellular pertussis (AP),diphtheria-tetanus-pertussis (DTP), or multicomponent acellular DTP(ADTP) toxoids. Adults Children Before immunization Postimmunization day28 Before immunization After immunization AP AP ADTP ADTP PlaceboCP_(10/5/5/3) Placebo CP_(10/5/3) DTP CP_(10/10/5/3)DT DTPCP_(10/10/5/3)DT Pertussis 16.45 22.78 16.56 415.87 43.71 15.45 221.32306.55 toxoid (9.46-28.62) (12.11-42.86) (9.08-30.22) (243.91-(14.29-133.88) (8.50-28.10) (99.83-490.67) (155.84- 709.09) 603.03)Filamentous 15.24 23.59 13.36 317.37 2.93 3.86 30.06 29.86 hemagglutinin(10.28-22.60) (15.59-35.69) (7.71-23.16) (243.05- (1.81-4.73)(3.03-4.93) (11.82-76.46) (16.51-53.99) 141.41) Agglutinogens 21.2628.64 27.0 2048.00 26.72 29.24 315.2 1243.3 (12.14-37.23) (12.20-67.21)(15.37-47.78) (1025.62- (16.94-42.15) (13.63-62.75) (127.4-779.9)(594.8-2603.5) 4089.55) Pertactin 7.89 11.47 7.46 855.13 6.54 9.45 60.13116.16 (4.00-15.56) (6.41-20.55) (3.51-15.87) (396.41- (2.79-15.33)(5.50-16.23) (24.59-147.04) (57.87-233.19) 1844.67) CHO cell 12.30 21.1110.78 604.67 27.47 9.71 270.60 342.51 neutralizing (6.97-21.68)(10.35-43.06) (5.54-20.97) (403.82- (7.36-102.62) (4.71-20.03)(24.6-1100.8) (146.6-800.2) assay 405.41) Diphtheria <0.1 <0.1 <0.1 <0.1<0.1 <0.1 8.75 9.65 toxoid (6.52-23.92) (5.62-16.57) Tetanus <0.1 <0.1<0.1 <0.1 <0.1 <0.1 4.11 6.32 toxoid (3.20-5.28) (5.31-7.53) No. studied16 15 16 15 10 25 12 25

[0193] TABLE 3 Serologic Results or Acellular Pertussis Vaccines InInfants (2, 4 and 6 Months Old) Geometric Mean Titres CHO Cell ClinicalNumber of Fimbrial Neutraliz- Trial Product Study Participants PT FHA 69kDa agglutinogens ation Agglutination Tet Dip 1 CP_(10/5/5)DT U.S. NIAID108 38 37 3 229 160 85 7.8 0.8 CP_(10/5/5/3)DT Multicentre 113 36 36 113241 150 73 5.0 0.4 Whole Cell (Mass.) Comparative Study 95 20 51 101 7080 42 − − Whole Cell (Lederle) (Cycle I) 312 67 3 64 193 270 84 − − 2CP_(10/5/5/3)DT Phase II 315 87.1 50.2 29.9 239.8 29.6 − 1.5 0.3 WholeCell (CLL) Canada 101 20 4.7 6.4 603.2 2.6 − 1.2 0.4 3 CP_(10/5/5/1)DTPhase IIB 32 58.4 45.2 40.6 111.4 32.7 − 1.0 0.14 CP_(20/20/5/1)DTCanada 33 133.3 95.0 37.1 203.8 82.4 1.1 0.21 Whole Cell (CLL) 30 10.48.9 6.8 393.9 4.0 1.8 0.31 4 CP_(10/5/5/1)DT Phase IIC 42 105.1 82.571.1 358.6 66.9 307.0 2.0 0.33 CP_(20/20/5/3)DT Canada 250 101.6 163.987.6 220.6 68.7 219.2 1.8 0.38 5 CP_(20/20/5/3)DT Montreal 58 212.7 83.4106.3 601.9 109.6 − 1.9 0.53 Whole Cell (CLL) Feasibility Study 58 101.411.7 16.8 906.9 6.0 1.1 0.27 6 CP_(10/5/5)DT U.S. NIAID 80 42 34 50 310196 185 CP_(20/20/5/3)DT Comparative Study 80 39 87 43 184 254 137 − −Whole Cell (CLI) (Cycle II) 80 2 3 9 33 54 167 Whole Cell (Lederle) 8018 2 16 129 137 86

[0194] TABLE 4 Efficacy of Acellular Pertussis Vaccines Efficacy %Vaccine A B CP_(10/5/5/3)DT 84.7 (80.3→88.5)¹ 77 PT₂₅.FHA₂₅DT 58(49.8→64.8)¹ DPT² 47.9 (37.1→56.9)¹

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What we claim is:
 1. A vaccine composition for protecting an at-riskhuman population against a case of disease caused by infection by B.pertussis, which comprises pertussis toxoid, filamentous haemagglutinin,pertactin and agglutinogens of B. pertussis in purified form in selectedrelative amounts to confer protection to the extent of at least about70% of members of the at-risk population.
 2. The vaccine of claim 1wherein said pertussis toxoid is present in an amount of about 5 toabout 30 μg nitrogen, said filamentous haemagglutinin is present in anamount of about 5 to about 30 μg nitrogen, said pertactin is present inan amount of about 3 to about 15 μg nitrogen and said agglutinogens arepresent in an amount of about 1 to about 10 μg nitrogen, in a singlehuman dose.
 3. The vaccine of claim 2 containing about 10 μg nitrogen ofpertussis toxoid, about 5 μg nitrogen of filamentous haemagglutinin,about 5 μg nitrogen of pertactin and about 3 μg nitrogen ofagglutinogens in a single human dose.
 4. The vaccine of claim 2containing about 20 μg nitrogen of pertussis toxoid, about 20 μgnitrogen of filamentous haemagglutinin, about 5 μg nitrogen of pertactinand about 3 μg nitrogen of agglutinogens in a single human dose.
 5. Thevaccine of claim 1 wherein the extent of protection is at least about80% for a case of pertussis having a spasmodic cough of duration atleast 21 days and confirmed bacterial infection.
 6. The vaccine of claim1 wherein the extent of protection is at least about 70% for a case ofmild pertussis having a cough of at least one day duration.
 7. Thevaccine of claim 2 wherein the extent of protection is about 85% for acase having a spasmodic cough of duration at least 21 days and confirmedbacterial infection.
 8. The vaccine of claim 1 wherein said agglutinogencomprise fimbrial agglutinogen 2 (Agg 2) and fimbrial agglutinogen 3(Agg 3) substantially free from agglutinogen
 1. 9. The vaccine of claim8 wherein the weight ratio of Agg 2 to Agg 3 is from about 1.5:1 toabout 2:1.
 10. The vaccine of claim 1 further comprising tetanus toxoidand diphtheria toxoid.
 11. The vaccine of claim 10 wherein saiddiphtheria toxoid is present in an amount of about 15 Lfs and tetanustoxoid is present In an amount of about 5 Lfs.
 12. The vaccine of claim1 further comprising an adjuvant.
 13. The vaccine of claim 12 whereinthe adjuvant is alum.
 14. A method of immunizing an at-risk humanpopulation against disease caused by infection by B. pertussis, whichcomprises administering to members of the at-risk human population animmunoeffective amount of the vaccine composition of claim 1 to conferprotection to the extent of at least about 70% of the members of theat-risk population.
 15. The use of purified forms of pertussis toxoid,filamentous haemagglutinin, pertactin and fimbrial agglutinogens of B.pertussis in the manufacture of a vaccine composition for administrationto an at-risk human population to confer protection to the extent of atleast about 70% of members of said at-risk human population.
 16. The useof claim 15 wherein there is used In the manufacture of a single humandose of the vaccine composition, from about 5 to about 30 μg of nitrogenof said pertussis toxoid, about 5 to about 30 μg of nitrogen.